The enormous increase in the demand for synthetic oligonucleotides, fueled by the advances in DNA technology, has been even further increased by recent progress in sequencing and decoding whole genomes, in particular the human genome. Many of the methods employed in molecular biology and DNA-based diagnostics to amplify, detect, analyze and quantify nucleic acids are dependent on chemically synthesized oligonucleotides. For instance, chemically synthesized oligonucleotides are employed in recombinant host-vector systems, which are used for techniques such as site-directed mutagenesis. They are also used in PCR methods, in which oligonucleotide primers are employed in temperature-cycled enzymatic amplifications of nucleic acids. Primers are also needed for state of the art sequencing techniques featuring enzymatic elongation and random termination. Another rapidly growing field is the application of oligonucleotides in hybridization assays, which are based on the specific annealing of oligonucleotide probes to the region of a nucleic acid analyte having a complementary sequence. Probes with covalently conjugated dyes that generate a fluorescent signal upon a perfect match hybridization are among the latest developments in this field. Corresponding methods to test for specific genomic epitopes, such as allelic discrimination or SNP detection employ hybridization probes and are readily multiplexable. Thus, automated high-throughput setups capable of simultaneously screening a vast number of analytes using miniaturized arrays of hybridization probes, such as DNA chips, are used for applications like genotyping and expression profiling. These and related methods will likely have an enormous impact in the areas of drug development and health management, among others.
Antisense technology is another field that demands a rapidly increasing supply of oligonucleotides. Synthetic antisense oligonucleotides are complementary to an RNA or DNA target sequence and are designed to halt a biological event, such as transcription, translation or splicing. They represent a whole new class of therapeutic agents, which have been shown to exhibit antiviral activity by inhibiting viral DNA or protein synthesis and, moreover, may be able to cure certain diseases by inhibiting gene expression via specifically recognizing and binding mRNA. The most recent generation of antisense oligonucleotides are comprised of backbone-modified DNA or RNA derivatives such as 2′-OMe-RNA, phoshorothioate, morpholino nucleic acid, LNA, or combinations thereof. In this manner, the properties of antisense compounds, such as nuclease resistance, RNase H susceptibility and binding affinity, which are crucial for either blocking or promoting the degradation of the complementary mRNA, can be modulated and optimized.
Another process of posttranscriptional, sequence-specific gene silencing is RNA interference (RNAi), which is presently being intensely investigated. RNAi is initiated inside cells by small interfering RNAs (siRNA), which are double-stranded RNA oligomers having 20 to 23 base pairs. The RNAi process has been utilized for functional analysis of mammalian genes and might also allow therapeutic applications.
The present state of the art for conducting automated oligonucleotide synthesis is the solid phase approach using phosphoramidite chemistry, which is based on the developments of Caruthers et al. (see e.g., McBride and Caruthers (1983) Tetrahedron Letters 24:245-248; Caruthers et al. U.S. Pat. Nos. 4,415,732 and 4,458,066) and Sinha et al. ((1983) Tetrahedron Letters 24:5843-5846) and which has been, together with related methods, such as the hydrogen-phosphonate chemistry, extensively reviewed by Beaucage and Iyer (1992) Tetrahedron 48:2223-2311. Each of these references is specifically incorporated herein by reference in its entirety.
During phosphoramidite mediated chemical oligonucleotide synthesis a series of nucleotide synthons are sequentially attached in a predetermined order to either, depending on the direction of chain extension, the 5′-functional group or the 3′-functional group of the growing strand, which is linked to a solid phase, such as CPG or a polystyrene resin. The attachment of each synthon generally comprises the following steps: 1) deprotecting the functional group of the growing strand, commonly the 5′-hydroxyl group; 2) coupling by adding a nucleoside synthon and an activator; 3) capping of unreacted terminal functional groups through introduction of an inert protecting group, to prevent further coupling to failure sequences; and 4) oxidizing the newly formed internucleosidic phosphorous linkage to the naturally occurring pentavalent state. This synthetic scheme, applying phosphoramidite chemistry, has reached a high level of optimization featuring routine stepwise coupling efficiencies of up to 99% on average per cycle. Following the completed assembly of the desired sequence the oligonucleotide is finally released and deprotected at the nucleobases usually in one step employing basic conditions.
In order to cope with the rapidly increasing demand for oligonucleotides, improvements in the currently available synthetic procedures are highly desirable, particularly to increase the number of oligonucleotides produced per day and per synthesizer. Promising approaches to enhance the throughput rate of oligonucleotides produced are methods that allow for the synthesis of not one, but two or more oligonucleotides in one synthetic run on a single batch of solid support. In this way, time and expense per oligonucleotide are greatly reduced due to lower amounts of support needed and lower numbers of samples that have to be handled, processed and stored. This strategy is especially favorable in cases in which all of the oligonucleotides produced in one synthetic run can be or even need to be added to the final application at the same time. This applies particularly to primer pairs as used for PCR or sequencing and for double stranded DNA or RNA probes. These combined oligonucleotide preparations also lead to a reduced number of samples to be stored and handled for the final application.
To date, prior art methods applying the strategy of the combined synthesis of oligonucleotides on the same support are all based upon the serial assembly at the same anchor moiety. The oligonucleotides are therefore, interconnected by cleavable linkers and thus are all simultaneously released in the final cleavage/deprotection step. For example, Hardy et al. (1994) Nucleic Acids Res. 22:2998-3004 and McLean et al. U.S. Pat. Nos. 5,393,877 and 5,552,535, each of which is specifically incorporated herein by reference in its entirety, have introduced the “TOPS” (two oligonucleotides per synthesis) methodology, which is based on a linker featuring a phosphoramidite moiety and a DMT group. In the course of the automated assembly using, possibly modified, standard procedures for coupling phosphoramidite monomers the linker can be integrated into the oligomeric chain partitioning it into the desired oligonucleotide units. One major drawback of the TOPS method is the harsh conditions and the long reaction times required to cleave the products from the linker (concentrated ammonia, 80° C., overnight; alternatively: conc. ammonia/40% aqueous methylamine (1:1, v/v), 60° C., 8 hours).
A modification of this technique is described by Pon et al. (WO 02/20541) and Holland et al., (WO 92/06103). Each of these references is specifically incorporated herein by reference in its entirety. Instead of the aforementioned separate linker amidite monomers, which are attached in a separate coupling cycle, in this method a linker unit is introduced as part of the modified nucleoside phosphoramidites. One of the oligonucleotides produced by this method necessarily carries a 5′-terminal phosphate group and is therefore not useful for some biochemical applications.
Each of the above-described methods for the tandem synthesis of oligonucleotides, include the coupling of specialty amidites, which is usually not very efficient and requires elongated coupling times or even repeated coupling. Another drawback of both methods is the need to prepare the specialty amidites. The synthesis of such linkers or modified nucleoside amidites is generally lengthy and time consuming. In addition, spare synthesizer ports are required on an automated nucleic acid synthesizer to introduce the specialty amidites into the synthetic process. Also, both methods result in an increasing number of truncated sequences and decreasing coupling efficiencies as the oligomer chain length progresses. Thus, the oligonucleotide units positioned closer to the end of the chain are of deteriorating quality and diminishing concentration. Thus, there remains a need for an improved method for the tandem synthesis of oligonucleotides.